8

Scanning Electron Microscopy: Principle,

Components and Applications
Dr. M. Kannan

Scanning Electron Microscope functions exactly as their optical counterparts

except that they use a focused beam of electrons instead of light to “image”
the specimen and gain information as to its structure and composition. Given
sufficient light, the unaided human eye can distinguish two points 0.2 mm apart.
If the points are closer together, they will appear as a single point. This distance
is called the resolving power or resolution of the eye. Similarly, light microscopes
use visible light (400- 700nm) and transparent lenses to see objects as small as
about one micrometer (one millionth of a meter), such as a red blood cell (7 μm) or
a human hair (100 μm). Light microscope has a magnification of about 1000x and
enables the eye to resolve objects separated by 200 nm. Electron Microscopes were
developed due to the limitations of light microscopes, which are limited by the
physics of light. Electron Microscopes are capable of much higher magnifications
and have a greater resolving power than a light microscope, allowing it to see
much smaller objects at sub cellular, molecular and atomic level. The smallest the
wavelength of the illuminating sources is the best resolution of the microscope.
De Broglie defined the wavelength of moving particles (electron) λ = h/mv, Where
λ= wavelength of particles, h= Planck,s constant, m= mass of the particle (electron),
v= velocity of the particles; after substituting the known values, λ = 12.3 Ao/V
The resolution of an optical microscope is defined as the shortest distance between
two points on a specimen that can still be distinguished by the observer or camera
system as separate entities. Resolution (r) = λ/ (2NA), Where λ is the imaging
wavelength, NA is objective numerical aperture.
Magnification is the process of enlarging the appearance, not physical size, of
something. Magnification is defined as the ratio of image distance versus object
distance
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M= v/u, Where M= magnification, u= object distance, v= image distance

Magnification is also defined as the ratio of the resolving power of the eye to
resolving power (δ) of the microscope M= δ eye/ δ microscope

Resolution Magnification

Difference between light microscope and electron microscope

Sl. Feature Light microscope Electron microscope

No.
1. Electromagnetic Visible light, 400- Electrons, app. 4nm
spectrum 700nm Monochrome
Colours visible
2. Maximum resolving app. 200nm 0.5nm with very fine
power detail
3. Maximum magnification x1000 to x1500 x500000
4. Radiation source Tungsten or quartz High voltage (50kV)
halogen lamp tungsten lamp,
Lanthanum hexaboride
5. Lenses Glass Electro magnetics
6. Interior Air-filled Vacuum
7. Focusing screen Human eye (retina), Fluorescent (TV) screen,
photographic film Photographic film
8. Preparation of Temporary mounts Tissues must be
specimens living or dead dehydrated = dead
9. Fixation Alcohol OsO4 or KMnO4
10. Embedding medium Wax Resin
11. Sectioning of specimen Hand or microtome Ultra microtome
sectioning < 20µm slice sectioning
Whole cells visible < 50nm slices
Parts of cells visible
12. Staining Water soluble dyes Heavy metals
13. Support for sample Glass slide Copper grid
Scanning Electron Microscopy: Principle, Components and Applications 83

Working principles of SEM

A beam of electrons is formed by the Electron Source and accelerated toward
the specimen using a positive electrical potential. The electron beam is confined
and focused using metal apertures and magnetic lenses into a thin, focused,
monochromatic beam. Electrons in the beam interact with the atoms of the
specimen, producing signals that contain information about its surface topography,
composition and other electrical properties. These interactions and effects are
detected and transformed into an image.
84 A Textbook on Fundamentals and Applications of Nanotechnology

Components of SEM
Electron Column
The electron column is where the electron beam is generated under vacuum,
focused to a small diameter, and scanned across the surface of a specimen by
electromagnetic deflection coils. The lower portion of the column is called the
specimen chamber.
Electron gun: An electron beam is thermionically emitted from an electron gun
fitted with a tungsten filament cathode. Tungsten has the highest melting point
and lowest vapour pressure of all metals, thereby allowing it to be heated for
electron emission, and because of its low cost. Other types of electron emitters
include lanthanum hexaboride (LaB6) cathodes, and field emission guns (FEG),
which may be of the cold-cathode type using tungsten single crystal emitters or
the thermally assisted Schottky type, using­emitters of zirconium oxide.
Condenser Lenses: After the beam passes the anode it is influenced by two
condenser lenses that cause the beam to converge and pass through a focal point.
In conjunction with the selected accelerating voltage the condenser lenses are
primarily responsible for determining the intensity of the electron beam when it
strikes the specimen.
Apertures: The function of these apertures is to reduce and exclude extraneous
electrons in the lenses. The final lens aperture located below the scanning coils
determines the diameter or spot size of the beam at the specimen. The spot size on
the specimen will in part determine the resolution and depth of field. Decreasing
the spot size will allow for an increase in resolution and depth of field with a loss
of brightness.
Scanning System: Images are formed by rastering the electron beam across the
specimen using deflection coils inside the objective lens. The stigmator or
astigmatism corrector is located in the objective lens and uses a magnetic field in
order to reduce aberrations of the electron beam. The electron beam should have a
circular cross section when it strikes the specimen however it is usually elliptical
thus the stigmator acts to control this problem.
Specimen Chamber: The lower portion of the column is specimen stage and
controls are located. Specimens are mounted and secured onto the stage which
is controlled by a goniometer. The secondary electrons from the specimen are
attracted to the detector by a positive charge Manual stage controls are found on
the front side of the specimen chamber for x-y-z movement.
Electron Detectors: Detectors collect the signal generated from interaction of beam
with specimen. Electronic detectors convert the signal into digital images and
most often collected signal are Secondary electrons by secondary electron detector
(Everhart–Thornley) Backscattered electrons by backscattered electrons detector
(Solid-State detector) and X-rays signal by Energy dispersive spectrometer (EDS)
detector.
Scanning Electron Microscopy: Principle, Components and Applications 85

Vacuum System: Vacuum is produced by an oil diffusion pump backed by a

mechanical pump. In the diffusion pump a stream of hot oil vapor strikes and
pushes air molecules toward a mechanical pump that expels them from the
system. A mechanical pump and valve system are used to preevacuate the system
because a diffusion pump only operates after a vacuum is created. If the column is
in a gas filled environment, electrons will be scattered collide with air molecules
which would lead to reduction of the beam intensity and stability. Similarly, other
gas molecules, which could come from the sample or the microscope itself, could
form compounds and condense on the sample. This would lower the contrast
and obscure detail in the image. The chemical and thermal stability is necessary
for a well-functioning filament (gun pressure). The field emission gun, LaB6 and
tungsten filament requires ~ 10-10, ~ 10-6 and 10-4 Torr, respectively. Hence, gun
column of electron microscope require vacuum to facilitate the electrons signals
from the sample to the detector for better imaging.
86 A Textbook on Fundamentals and Applications of Nanotechnology

How Scanning Electron Microscope (SEM) works

Ernst Ruska and Max Knoll developed first electron microscope during 1931with
resolution of 100nm and later by addition of electromagnetic lenses, brought the
resolution to 0.05nm. SEM is similar to the optical stereo-binocular microscope to
observe the morphology and shape of the specimen.
¾¾ The electron gun produces an electron beam when tungsten wire is heated
by current and accelerated by the anode.
¾¾ The beam travels in the vacuum column through electromagnetic fields
and lenses, which focus the beam down toward the sample.
¾¾ A mechanism of deflection coils enables to guide the beam so that it scans
the surface of the sample in a raster pattern.
¾¾ When the incident beam touches the surface of the sample and produces
signals viz.,
yy Secondary electrons (SE)
yy Auger electrons
yy Back scattered electrons (BSE)
yy Characteristic X – Rays
yy Cathodoluminescence
¾¾ The emitted signals are trapped by electrical detectors, convert into digital
images and displayed on a screen as digital image.
¾¾ Provides information sample’s elemental composition, structural variation
and morphology.
¾¾ In the SEM, use much lower accelerating voltages to prevent beam
penetration into the sample since the requirement is generation of the
secondary electrons from the true surface structure of a sample. Therefore,
it is common to use low KV, in the range 1-5kV for biological samples, even
though the SEMs are capable of up to 30 kV.
Scanning Electron Microscopy: Principle, Components and Applications 87

Interaction of Electron Beam with Specimen:

When the primary electron beam interacts with the sample, the electrons lose
energy by repeated random scattering and absorption within a teardrop-shaped
volume of the specimen known as the interaction volume, which extends from less
than 100 nm to approximately 10 µm into the surface. The size of the interaction
volume depends on the electron’s landing energy, the atomic number of the
specimen and the specimen’s density. The energy exchange between the electron
beam and the sample results in the reflection of high-energy back scattered
electrons by elastic scattering, emission of low energy secondary, auger electrons
by inelastic scattering and the emission of electromagnetic radiation (X-rays and
cathodoluminescence), each of which can be detected by respective detectors. The
beam current absorbed by the specimen can also be detected and used to create
images of the distribution of specimen current. Electronic amplifiers of various
types are used to amplify the signals, electronic detectors convert the signals into
digital images and displayed on a computer monitor.

Backscattered electron: Those electrons, which are deflected, back in the direction
of the beam. The special detector in scanning and transmission electron
88 A Textbook on Fundamentals and Applications of Nanotechnology

microscope traps these signals. These are used to discriminate areas of different
atomic numbered elements. Higher atomic numbered elements gives off more
backscattered electrons and appear brighter than lower numbered elements. It has
the resolution to the level of 1000 nm. These electrons have high energy.
Secondary Electrons: These electrons are also collected with a special type of
detector used in SEM and TEM. They are used primarily to reveal topographical
feature of a specimen. It has the resolving power <10 nm. These electrons have
low energy.
Auger Electrons: These are special types of low energy electrons that carry the
information about the chemical nature (atomic composition) of the specimen.
These are generated from the upper layer of specimen. It is a powerful tool in
the material sciences for studying the distribution of the lighter numbered
atomic elements on the surface of the specimen. It has limited application in
biological sciences. It is specialized equipment known as scanning auger
electron spectrometer.
Cathodoluminescent: This effect results when the energy of the impinging
electrons in converted into visible light. Certain types of compounds are capable
of cathode luminescence and detected by special types of detector. The resolution is
the similar to the light microscope.
Bremsstrahlung: Two important types of x-ray may be generated when the beam
electron encounters the atoms of the specimen, continuous or bremsstrahlung
x-ray and characteristic x-ray are generated when incoming, beam passing
close to the atomic nucleus is slowed by the coulomb field of the nucleus with
the release of x- ray energy. The intensity of x-ray energy released depends
on how close the electron comes to the nucleus closer. The closer passes
decelerate the electron more and yield higher energy x-rays. These are used
to measure specimen mass thickness when quantitative analysis performed on
thin sections. These are continuous x-rays also known as background or white
radiation.
Characteristic X-rays: When high energy beam electrons interact with the
shell electrons of the specimen atoms so that an inner shell electron is ejected.
The removal of this electron temporarily ionizes the atom until an outer shell
electron drops into the vacancy to stabilize the atom. Since this electron comes
from a higher energy level, a certain amount of energy must be given off before
it will be accommodated in the inner shell. The energy is released as an x-ray,
the energy which equals the difference in energy between the two shells. Since
this x-ray is of a discrete energy level, rather than a continuous, this event
may be plotted as discrete peaks. Different elements will fill the vacancies
in shells in unique ways. This means that since each element will generate a
unique series of peaks, the spectrum may be used to identify the elements; such
discrete x-rays are termed characteristic x-rays. The equipment for detection
x-rays are energy dispersive x-ray (EDX) detector and Wavelength Dispersive
X-ray (WDX) Detector.
Scanning Electron Microscopy: Principle, Components and Applications 89

Sample preparation for SEM

Step Chemical Temperature Time Repetitions

Primary 2.5% room or 0-4°C 2-4 hours or 1
fixation glutaraldehyde in microwave
distilled water
Wash distilled water room or 0-4°C 30 minutes 3-5
Secondary 1-4% osmium room or 0-4°C 2-4 hours 1
fixation tetroxide in
distilled water
Wash distilled water room or 0-4°C 30 minutes 3-5
Dehydration 25% ethanol room or 0-4°C 20 minutes 1
50% ethanol 20 minutes 1
70-75% ethanol 20 minutes 1
90-95% ethanol 20 minutes 1
100% ethanol 30 minutes 2
Critical point dry
Mount on specimen stub with silver paste or graphite
Sputtering coat the biological sample with gold/palladium alloy for making them
conductive
Store stubs in desiccator and view the external surface with SEM

SEM micrographs
90 A Textbook on Fundamentals and Applications of Nanotechnology

Applications of Scanning Electron Microscopy

Topography: The surface features of an object or “how it looks”, its texture; direct
relation between these features and materials properties (hardness, reflectivity...
etc.)
Morphology: The shape and size of the particles making up the object; direct
relation between these structures and materials properties (ductility, strength,
reactivity...etc.)
Composition: The elements and compounds that the object is composed of and the
relative amounts of them; direct relationship between composition and materials
properties (melting point, reactivity, hardness...etc.)
Crystallographic Information: How the atoms are arranged in the object; direct
relation between these arrangements and materials properties (conductivity,
electrical properties, strength.etc.)

Advantages of SEM
¾¾ It gives detailed 3D and topographical imaging and the versatile
information garnered from different detectors.
¾¾ This instrument works very fast.
¾¾ Modern SEMs allow for the generation of data in digital form.
¾¾ Most SEM samples require minimal preparation actions.
Disadvantages of SEM
¾¾ SEMs are expensive and large.
¾¾ Special training is required to operate an SEM.
¾¾ The preparation of samples can result in artifacts.
¾¾ SEMs are limited to solid samples.
¾¾ SEMs carry a small risk of radiation exposure associated with the electrons
that scatter from beneath the sample surface.

References
Goldstein, J.I., Yakowitz, H.. Newbury, D.E Lifshin, E.. Colby, J.W Colby J.W. and. J.R.
Coleman. 1975. Pratical Scanning Electron Microscopy: Electron and Ion
Microprobe Analysis.
Loretto, M.H. 1984. Electron Beam Analysis of Materials, in Chapman and Hall,
London New York FEI. The Quanta 200 User’s Operation Manual 2nd
ed. (2004). I.M. Watt, The Principles and Practice of Electron Microscopy,
(Cambridge Univ. Press. Cambridge, England, 1985.
Lyman, C.E., Newbury, D.E. Goldstein, J.I. Williams, D.B. Romig, A.D. Armstrong,
J.T. Echlin, P.. Fiori, C.E Joy, D.C. Lifshin E.and Klaus-Ruediger Peters,
Scanning Electron Microscopy: Principle, Components and Applications 91

1 9 9 0 Scanning Electron Microscopy X-Ray Microanalysis and Analytical

Electron Microscopy: A Laboratory Workbook, Press. New York, N.Y.
Postek, M.T.,. Howard, K.S Johnson A.H. McMichael K.L.1980. Scanning Electron
Microscopy: A Student’s Handbook, (Ladd Research Ind., Inc. Williston,
VT.).

Questions
Fill in the blanks:
1. Electron microscope uses ................... as a source for making images.
2. Electron microscope was invented by ...................
3. Resolution of unaided human eye is ...................
4. Primary fixative used in sample preparation of SEM is ...................
5. Formula for Resolution is ...................
Choose the correct answer
1. What is the resolving power of light microscope?
ii) 200 �m iii) 0.02m
iv) 200nm v) 0.2 mm
2. Which of the following is the first step in the processing of biological
material for transmission electron microscopy?
i) Dehydration ii) Sectioning
iii) Fixation iv) Embedding
3. A vacuum is needed in the electron microscope to.........................................

i) Pull the electrons onto the ii) Eliminate molecules of nitrogen,

specimen oxygen or carbon dioxide
iii) Pull the specimen into the iv) Prevent secondary radiation
column affecting the microscope control
panel
4. Which of the following statements about SEM is true?
i) The specimen is usually ii) Carbon nanotubes
coated with gold
iii) Quantum dots iv) All the above

5. Ernst Ruska awarded Nobel prize during 1986 for their invention of______
i) SEM ii) TEM
iii) STM iv) AFM
92 A Textbook on Fundamentals and Applications of Nanotechnology

True or False
1. Secondary electrons are formed by collision of incident beam and sample
2. In SEM copper grid is used as platform for sample analysis
3. Electron microscope was invented in the year 1931 by Max Knoll and
Ernst Ruska.
4. In electron microscopy, the lenses used to magnify the image are made of
glasses
5. 2.5% glutaraldehyde is used as primary fixative for SEM sample
preparation
Short notes
1. What is meant by backscattered electron
2. Light vs electron microscope differentiate
3. Why vacuum is needed in electron microscope?
4. Narrate the role of different components of SEM with illustration
5. Advantage and disadvantage of SEM?
Essay
1. Write in detail about essential components and working principle of
scanning electron microscope with diagram